Validation of isoelectric focusing method for recombinant erythropoietin detection in human urine
DOI:
https://doi.org/10.25758/may.1194Keywords:
Erythropoietin, Isoelectric focusing, Double blot, Chemiluminescent detection, Method validationAbstract
Erythropoietin (EPO) is a substance that stimulates red blood cell production, increasing muscle oxygenation, and is secreted naturally by the body and excreted in the urine in low concentrations. Due to the special properties of EPO, this was quickly introduced into the world of sports and its illicit use provides advantages in sports performance. In early 2000, was developed a method for direct detection of erythropoietin (EPO) on recombinant human urine by Lasne, based on isoelectric focusing (IEF) in polyacrylamide gel, followed by double blotting, has been published and validated. In 2002, the World Anti-Doping Agency (WADA) implemented this same method which is currently the only official method used by laboratories accredited by WADA. The starting point for this work was the need to implement and validate the reference method, for the detection of recombinant erythropoietin in human urine. The study was conducted at the Laboratory for Doping Analysis (LAD) of the Sports Institute of Portugal (IDP), current IPDJ. The objective of the work focused on validation studies/investigation of different validation parameters (specificity/selectivity; ability identification, detection limit, accuracy, and repeatability), according to the protocol Procedimento Geral Interno of the Antidoping Laboratory of Lisbon. This method of screening and confirmation has revealed performance characteristics in accordance with the applicable requirements for what is considered valid and fit.
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